afimoxifene and Melanoma

Recent reports suggest that N-methyl-d-aspartate receptor (NMDAR) blockade by MK-801 decreases tumor growth. Thus, we investigated whether other ionotropic glutamate receptor (iGluR) antagonists were also able to modulate the proliferation of melanoma cells. On the other hand, the antiestrogen tamoxifen (TAM) decreases the proliferation of melanoma cells, and is included in combined therapies for melanoma. As the efficacy of TAM is limited by its metabolism, we investigated the effects of the NMDAR antagonist MK-801 in combination with TAM and its active metabolites, 4-hydroxytamoxifen (OHTAM) and endoxifen (EDX). The NMDAR blockers MK-801 and memantine decreased mouse melanoma K1735-M2 cell proliferation. In contrast, the NMDAR competitive antagonist APV and the AMPA and kainate receptor antagonist NBQX did not affect cell proliferation, suggesting that among the iGluR antagonists only the NMDAR channel blockers inhibit melanoma cell proliferation. The combination of antiestrogens with MK-801 potentiated their individual effects on cell biomass due to diminished cell proliferation, since it decreased the cell number and DNA synthesis without increasing cell death. Importantly, TAM metabolites combined with MK-801 promoted cell cycle arrest in G1. Therefore, the data obtained suggest that the activity of MK-801 and antiestrogens in K1735-M2 cells is greatly enhanced when used in combination.

Topics: Animals; Antineoplastic Agents, Hormonal; Cell Proliferation; Dizocilpine Maleate; Drug Evaluation, Preclinical; Drug Therapy, Combination; Excitatory Amino Acid Antagonists; Melanoma; Mice; Tamoxifen; Tumor Cells, Cultured

Due to elaborate control mechanisms, in benign tumors the activation of oncogenes primarily induces senescence, associated with cessation of cellular proliferation; for example, melanocytic nevi expressing mutant B-Raf. These mechanisms include the RB and/or the p53 pathway. The current model of melanomagenesis postulates that progression to immortal melanoma cells requires inactivating aberrations in signaling cascades controlling senescence. Thus, melanoma cells carrying mutant B-Raf should be resistant to mitogen-activated protein kinase (MAPK) pathway-induced senescence. Here, we demonstrate that hyperactivation of the MAPK pathway following activation of an inducible form of oncogenic C-Raf induces a senescence-like proliferation arrest in B-Raf mutant melanoma cells. This Raf-induced senescence is initially strictly dependent on MEK signaling, but seems to be independent of MAPK signaling after prolonged continuance. It is associated with reduced levels of RB phosphorylation and an increase in p21 expression, but is independent of p16(Ink4a) and p53. These data argue against the existence of fundamental changes in melanoma cells completely precluding senescence.

Topics: Animals; Cell Division; Cell Line, Tumor; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p16; Estrogen Antagonists; Extracellular Signal-Regulated MAP Kinases; G1 Phase; Humans; MAP Kinase Signaling System; Melanoma; Mice; NIH 3T3 Cells; Phosphorylation; Proto-Oncogene Proteins B-raf; Receptors, Estrogen; Retinoblastoma Protein; Skin Neoplasms; Tamoxifen; Transduction, Genetic

The literature concerning cytoskeletal changes and metastatic progression is unresolved, some studies suggesting a positive association between the ability of cells to organize their cytoskeleton and others suggesting an inverse correlation. In an attempt to learn more about cytoskeletal changes and the ability of melanoma cells to interact with extracellular matrix proteins we examined the effects of pharmacological manipulation of cell attachment and cell invasion through fibronectin on levels of F-actin and vimentin in a highly metastatic cutaneous melanoma cell line (A375-SM cells). Additionally, we examined whether any correlation existed between the levels of the cytoskeletal proteins and subpopulations of the cell line of varying invasive ability. We report that agents which reduced cell attachment to plastic and invasion through fibronectin in vitro (tamoxifen, N-desmethyltamoxifen and 17beta-oestradiol) caused increases in levels of F-actin and vimentin, whereas agents which did not affect attachment or invasion (4-hydroxytamoxifen and dihydrotestosterone) had little or no effect on the cytoskeletal proteins. In contrast, however, those cells which were most effective at invading through fibronectin were significantly better at acutely increasing their levels of F-actin and vimentin than less invasive cells. We speculate that the ability to rapidly and possibly reversibly alter the cytoskeleton might be associated with metastatically successful cells in vivo.

Topics: Actins; Antineoplastic Agents, Hormonal; Cell Adhesion; Cytoskeletal Proteins; Cytoskeleton; Dihydrotestosterone; Embryonal Carcinoma Stem Cells; Estradiol; Fibronectins; Gene Expression Regulation, Neoplastic; Gonadal Steroid Hormones; Humans; Melanocytes; Melanoma; Neoplasm Invasiveness; Neoplasm Proteins; Neoplastic Stem Cells; Skin Neoplasms; Tamoxifen; Tumor Cells, Cultured; Vimentin

In this study we provide evidence that tamoxifen, the widely used breast cancer drug, is a potent antagonist of glycolipid metabolism. When added to the medium of cultured multidrug resistant (MDR) KB-V-1 carcinoma cells, tamoxifen, at 5.0 microM, drastically lowered the levels of glucosylceramide (glc-cer), as evidenced by a reduction in glc-cer mass. In a similar fashion, in cultured human melanoma cells grown with [3H]galactose, tamoxifen inhibited formation of glc-cer by 44%, and retarded lactosylceramide and ganglioside formation by 50 and 35%, respectively. When glc-cer synthase of melanoma was assayed in cell-free incubations, the inclusion of tamoxifen, at a 1:10 molar ratio with ceramide, inhibited glc-cer synthesis by 50%. These results clearly reveal a new action of tamoxifen and thereby pose intriguing questions regarding mechanisms of action in the realm of estrogen receptor-independent modalities, including effects on MDR.

Topics: Antineoplastic Agents, Hormonal; Chromatography, Thin Layer; Drug Resistance, Multiple; Glucosylceramides; Glucosyltransferases; Glycosphingolipids; Glycosylation; Humans; KB Cells; Melanoma; Molecular Structure; Neoplasms; Tamoxifen; Tumor Cells, Cultured

The triphenylethylene antiestrogen tamoxifen (TAM) is believed to exert its antitumor effect via the estrogen receptor (ER). To test this hypothesis and to differentiate between ER-mediated and general cytotoxic effects of TAM, the growth-inhibitory effects of TAM and its in vivo metabolite 4-hydroxytamoxifen (OH-TAM) have been studied in five continuous human cancer cell lines, MCF7 and T47D (mammary carcinoma, ER positive), BT20 and MDA-MB-231 (mammary carcinoma, ER negative), and ME8 (melanoma, ER negative). All five cell lines are completely killed by concentrations of TAM and OH-TAM above 10(-6) M, regardless of ER status. TAM and OH-TAM have little effect on the ER-negative lines at concentrations below 10(-6) M, whereas the ER-positive lines are highly sensitive to TAM at 10(-7) M and to OH-TAM at 10(-9) M. Inhibition of growth parallels the relative affinity of these drugs for the ER. We conclude that, above 10(-6) M, the growth-inhibitory effects of TAM and OH-TAM in tissue culture are the results of a mechanism other than that via the ER system and that only at lower concentrations are the true ER-mediated effects seen. Plasma concentrations of TAM and OH-TAM in breast cancer patients treated with TAM are in the same range as the concentrations in vivo at which growth inhibition is seen, leading to the conclusion that both compounds contribute to the overall effect of TAM in vivo.

Topics: Binding, Competitive; Breast Neoplasms; Cell Line; Cell Survival; Drug Evaluation, Preclinical; Estradiol; Estrogen Antagonists; Female; Humans; Kinetics; Melanoma; Receptors, Estrogen; Tamoxifen